After significant progress in the technology of gene therapy, the concept of using gene therapy to cure or alleviate inherited and acquired diseases has been accepted. Investigators have accomplished the requisite first steps: it has been shown that transferred genes can be induced to function in the human body. So far, however, no approach has definitively improved the health of one of the more than 2,000 patients who have enrolled in gene therapy trials worldwide. This lack of a convincing therapeutic benefit may reflect researchers' imperfect initial groping toward a difficult new technology and that the obstacles are more formidable than expected.
An individual gene in the human cell is a stretch of DNA that, in most cases, acts as a blueprint for making a specific protein. All cells in a body carry the same genes in the chromosomes of the nucleus. But different cells use, or express distinct subsets of genes and hence make separate sets of proteins.
The regulation of the difference in gene expression, according to the cell type (and cell needs), is controlled by different DNA fragment named promoter and/or enhancer in different cells. Only with the promoter, and sometimes together with enhancer, will genes be expressed. The gene expression is a two step process. The first is transcription that generates RNA from DNA template, and the second is translation that synthesizes protein from RNA template.
After completion of the protein synthesis, the proteins will be transported from their manufacture sites to their destined functional sites. There are two pathways for proteins to export from the cells where they are synthesized. The first pathway is constitutive secretion. This pathway presents a mechanism for bulk flow of proteins and lipids. The second pathway is a regulated process named exocytosis. In this pathway, a protein will only be released upon receiving a specific triggering signal, for instance, triggered by a hormone.
The mechanisms of natural protein synthesis and exportation are the main reasons that the primary research effort in gene therapy in the past decades and present has been focused on tissue or cell specific approaches. Researchers have been searching for tissue specific genes and methods to deliver them into their target organs or tissue, where the proteins are synthesized naturally, and the native proteins are exported and delivered to their functional sites through native pathways. For instance, naturally haemophilia factor XIII and human serum albumin are produced in liver cells and then exported into blood stream for their functions. Insulin is produced in pancreas beta-cells and also exported into blood stream for its function.
Although specific delivery of therapeutic genes into their native manufacturing sites is the ideal scheme for gene therapy, it is difficult to practice either in vivo or in vitro. In vitro, it is almost impossible to harvest enough target cells to introduce therapeutic genes into them. In turn, harvested target cells are not able to synthesize sufficient amount of proteins for the purpose of therapy. In vivo, a therapeutic gene has an equivalent probability entering any type of cells, regardless whether they are in the form of retroviral vector, liposome harbored retroviral vector or naked DNA. Consequently few of the DNA (therapeutic gene) will be delivered into its target cells. Therefore, the success of the current gene therapy approach critically depends on whether a tissue specific gene can be delivered specifically to the target organ or tissue. Despite tremendous effort and investment, the tissue specific approach has been found extremely difficult and unsuccessful so far.
On the other hand, non-tissue specific approaches also encounter serious obstacles. Although many cell types in the body are easy to obtain, such as muscle or skin cells, there are disadvantages with using non-tissue specific cells as host cells for gene therapy. In some cases, the host cells naturally do not posses the above described two protein export mechanisms. In other cases, even if the host cells do posses these export mechanisms for their native proteins, they do not function equivalently in the export of a non-native or guest protein. Low efficiency in exporting guest protein or complete blockage of the native export pathway has been reported.
Recently, gene therapy has achieved major progress by utilizing the native ability of viruses to enter cells, bringing their own genetic material with them. Many of these organisms have now been engineered to serve as vectors, or delivery vehicles, for gene transfer. Among variety of viruses, retroviruses are the most promising gene-delivery systems studied so far (Friedmann, Scientific American, June 1997, 96). Retroviruses convert their RNA to DNA in infected cells and insinuate the DNA into a chromosome. The integrated DNA then directs the synthesis of viral proteins.
Under normal circumstances, integrated retroviral DNA would direct the synthesis of viral proteins and RNA, which would then assemble into clones of the original virus. A method has been developed to alter retrovirus. The altered retrovirus, bereft of instructions for making viral proteins, produces no progeny. The virus essentially disappears from the cell, leaving behind only the foreign gene and nucleotide sequences that now serve merely to facilitate the expression of the gene.
U.S. Pat. No. 5,399,346 (to Anderson et al.) discloses a process for providing a human with a therapeutic protein. The process comprises inserting a DNA segment encoding a therapeutic protein into primary human cells, and introducing the primary human cells into a human. The primary human cells express and secret therapeutic protein in vivo. Anderson et al. further disclose that the primary human cells are nucleated blood cells, as well as progenitor and precursors thereof, which are capable of expanded growth in culture. Moreover, Anderson et al. teach that the genetically engineered cells can be combined with a pharmaceutically acceptable carrier for suitable administration to the human body. The carrier may be a liquid (e.g., a saline solution) or a solid carrier, e.g., an implant. In employing a liquid carrier, the engineered cells may be introduced, e.g., intravenously, sub-cutaneously, intramuscularly, intraperitoneally, intralesionaly, etc. Anderson et al. teach that the function of the therapeutic protein resides in the natural or modified function of the primary human cells in vivo.
One of the earliest gene therapies for curing human diseases was to use genetic engineered hemoglobin for treating beta thalassemia, a disorder of hemoglobin. Red blood cells of patients having beta thalassemia are deficient in beta globin, which in healthy individuals combines with alpha globin and heme to yield hemoglobin. The lack of beta globin gives rise a deficit in hemoglobin production and an excess of alpha globin, which in turn cause severe anemia. Researchers used beta globin promoter and beta globin gene (native to the red blood cells) to produce the desired beta globin. In these types of applications, the hemoglobin promoter has not been utilized in genetic engineering of heterologous proteins (non-native to the red blood cells) in red blood cells.
On the other hand, variety of native promoters have been used in constructing vectors carrying genes encoding therapeutic proteins that are either native or heterologous to the host cells. However, because of the concentrated effort in tissue specific gene therapy approach, the constructed vectors are essentially focused on the nucleated cells which have surface markers for specific target delivery of the cells.
Recently, various nonviral methods for therapeutic gene transfer have also been developed. For instance, liposomes have been used to harbor a retroviral vector (a stable loop of DNA derived from bacterial viruses known as phages) in which original genes have been replaced by those intended to be therapeutic. Injection of naked DNA (without lipid wrapping) into patients has also been explored. The naked DNA, injected into the muscle of an animal, was expressed as protein and a quite high local concentration of protein was obtained (Feigner, Scientific American, June 1997, 102). However, the local high concentration of protein produced inside the muscle would not have been sufficient to be effective against diseases like diabetes or hemophilias when the proteins are diluted into the three liters of plasma contained in the blood stream.
It is apparent that there is a strong need for new strategies and methods to overcome difficulties in gene therapy and to achieve the goal of synthesis and delivery of a sufficient amount of proteins in vivo.